Project Summary I am currently a senior level postdoctoral fellow with a primary research focus in opioid receptor pharmacology. I intend to transition to a junior faculty position in the next two years, and my current mentored position has been structured to accomplish this goal. I have a wide-range of experience with in vivo models of pain and addiction, as well as extensive experience with techniques to assess receptor binding, function, and localization ex vivo. I am currently working in the laboratory of Dr. Christopher Evans, a prominent opioid researcher, at the University of California Los Angeles (UCLA). During my time at UCLA, I will enhance my skill set by learning new techniques in primary cell culture, electrophysiology, and arrays. My mentor and I have also planned several strategies to facilitate my transition to a faculty position, including budgetary responsibility, teaching, and course work. UCLA is a renowned institution with a number of high profile researchers, and during this mentored phase I will develop important collaborations that I hope to sustain into my independence. In addition, the university also has several state-of-the-art core facilities through which I will enhance my research opportunities. My research focus is on the in vivo consequences of ligand directed signaling at the delta (?) opioid receptor. Agonists for this receptor are being developed for clinical use, as activation of ? receptors relieves pain, and reduces anxiety and depression. However, a major limitation to the clinical use of these compounds is that a subset of ? agonists also produce convulsions, the mechanism of which is unknown. This agonist-selective behavior could be due to differential trafficking and signaling of the ? receptor, and my recent work shows that the internalizing agonist, SNC80, produces convulsions which are not observed with the non-internalizing agonist, ARM390. -arrestins are major mediators of receptor internalization, and also mediate subsequent receptor signaling. One of the aims of this proposal is to determine the role of -arrestins in SNC80 and ARM390-induced behaviors, using -arrestin 1 and 2 knockout mice. In addition, to characterize the different signaling mechanisms regulating this functional selectivity, the distinct signaling complexes formed by SNC80 and ARM390 will be determined using proximity ligation assays and phosphoprotein arrays. Furthermore, I have found that repeated use of these agonists in an inflammatory pain model also results in differential tolerance. SNC80 produces receptor downregulation and generalized tolerance to all agonist-induced effects. In contrast, ARM390-tolerant animals show intact ? receptors, and analgesic tolerance only - the mechanism of which is unknown. A further aim of this application is to characterize this differential tolerance, by looking for changes in receptor-ion channel coupling within the dorsal root ganglia following chronic use. I will also examine the role of -arrestins in these two types of tolerance, and explore possible mechanisms underlying these differences using DNA microarrays. This work has important therapeutic implications, and will enhance our understanding of in vivo opioid receptor trafficking and signaling.